Project Summary/Abstract: LncRNA Regulators of Epidermal Homeostasis and Early Neoplasia Extensive regulatory mechanisms control cell growth and differentiation, the deregulation of which can lead to tumorigenesis. Investigation of the factors that regulate normal cellular homeostasis has revealed important insights in the understanding of neoplasia. Research on homeostatic regulators has primarily focused on protein coding genes, however long non-coding RNAs (lncRNAs) have emerged as critical factors in the control of proliferation and differentiation. Despite the growing number of functional lncRNAs, the majority have not been thoroughly investigated for their potential role in tumorigenesis. To identify novel lncRNAs involved in the regulation of epidermal homeostasis and early neoplasia, 15 human cutaneous SCC tumors and patient/site matched normal skin samples were subjected to RNA sequencing. Following stringent filtering criteria, including high expression, differential expression in SCC versus normal skin, and low protein coding potential, computational analyses identified 11 novel unannotated transcripts that are likely lncRNAs. These candidate lncRNAs were termed SCC Mis- Regulated Transcripts (SMRTs). Functional evaluation of one of these SMRTs, SMRT-2, which is downregulated in SCC, has revealed an essential role in epidermal differentiation. Thus, to our knowledge, SMRT-2 is the first identified lncRNA with an essential function in epidermal homeostasis that is mis-regulated in SCC. Given these findings, the goal of the work proposed here is to mechanistically characterize SMRT-2 to address how it influences epidermal homeostasis and how this is deregulated in early neoplasia. Specifically, in Aim I we will define the protein, RNA, and DNA interactors of SMRT-2 using the unbiased and high-throughput approaches of RNA-Protein Microarray Analysis (RNA-PMA), RNA Interactome Analysis sequencing (RIA-seq), and chromatin isolation by RNA purification sequencing (ChIRP-seq). Because the remaining ten SMRTs have not yet been studied for functionality, another goal of this work that will be addressed in Aim II is to functionally evaluate the remaining SMRTs by manipulating their expression levels in robust models of epidermal homeostasis: in vitro three dimensional epidermal tissue culture and in vivo skin xenografting onto immune deficient mice. Identified functionally active SMRTs will then be mechanistically characterized similarly to SMRT-2. Overall, these studies will provide insight into the role of lncRNAs in epidermal homeostasis and early neoplasia and identify additional factors essential for their function, potentially informing methods of cancer prevention, detection, and treatment.